Isolation, identification, and screening of biosurfactant-producing and hydrocarbon-degrading bacteria from oil and gas industrial waste.

Qatar is one of the biggest oil and gas producers in the world, coupled with it is challenging environmental conditions (high average temperature: >40 °C, low annual rainfall: 46.71 mm, and high annual evaporation rate: 2200 mm) harbors diverse microbial communities that are novel and robust, with the potential to biodegrade hydrocarbons. In this study, we collected hydrocarbon contaminated sludge, wastewater and soil samples from oil and gas industries in Qatar. Twenty-six bacterial strains were isolated in the laboratory from these samples using high saline conditions and crude oil as the sole carbon source. A total of 15 different bacterial genera were identified in our study that have not been widely reported in the literature or studied for their usage in the biodegradation of hydrocarbons. Interestingly, some of the bacteria that were identified belonged to the same genus however, demonstrated variable growth rates and biosurfactant production. This indicates the possibility of niche specialization and specific evolution to acquire competitive traits for better survival. The most potent strain EXS14, identified as Marinobacter sp., showed the highest growth rate in the oil-containing medium as well as the highest biosurfactant production. When this strain was further tested for biodegradation of hydrocarbons, the results showed that it was able to degrade 90 to 100% of low and medium molecular weight hydrocarbons and 60 to 80% of high molecular weight (C35 to C50) hydrocarbons. This study offers many promising leads for future studies of microbial species and their application for the treatment of hydrocarbon contaminated wastewater and soil in the region and in other areas with similar environmental conditions.

Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

Monitoring of anti-malarial drug resistance is vital in Northeast India as this region shares its international border with Southeast Asia. Genetic diversity of Plasmodium parasites regulates transmission dynamics, disease severity and vaccine efficacy. P. falciparum chloroquine resistance transporter (Pfcrt), multidrug resistance-1 (Pfmdr-1) and kelch 13 propeller (PfK-13) genes which govern antimalarial drug resistance and three genetic diversity markers, merozoite surface protein 1 and 2 (Pfmsp-1, Pfmsp-2) and glutamate rich protein (Pfglurp) were evaluated from Tripura, Northeast India using molecular tools. In the Pfcrt gene, 87% isolates showed triple mutations at codons M74I, N75E and K76T. 12.5% isolates in Pfmdr-1 gene showed mutation at N86Y. No polymorphism in PfK-13 propeller was found. Polyclonal infections were observed in 53.85% isolates and more commonly in adults (p = 0.0494). In the Pfmsp-1 locus, the K1 allelic family was predominant (71.2%) followed by the 3D7/IC family (69.2%) in the Pfmsp-2 locus. RII region of Pfglurp exhibited nine alleles with expected heterozygosity of 0.85. The multiplicity of infection for Pfmsp-1, Pfmsp-2 and Pfglurp were 1.56, 1.31 and 1.06 respectively. Overall, the study demonstrated a high level of chloroquine resistance and extensive parasite diversity in the region, necessitating regular surveillance in this population group.

Emergence of Multidrug- and Pandrug- Resistant Pseudomonas aeruginosa from Five Hospitals in Qatar.

BACKGROUND: A global rise in multidrug-resistant (MDR) nosocomial infections has led to a significant increase in morbidity and mortality. MDR Gram-negative bacteria (GNB) are recognised for rapidly developing drug resistance. Despite Pseudomonas aeruginosa being the second most common GNB isolated from healthcare associated infections, the magnitude of MDR P. aeruginosa (MDR-PA) has not been evaluated in Qatar. AIM: To assess the prevalence and antimicrobial susceptibility patterns of MDR-PA from 5 major hospitals in Qatar. METHODS: A total of 2533 P. aeruginosa clinical isolates were collected over a one-year period. MDR-PA was defined as resistance to at least one agent of ≥ 3 antibiotic classes. Clinical and demographic data were collected prospectively. FINDINGS: The overall prevalence of MDR-PA isolates was 8.1% (205/2533); the majority of isolates were from patients exposed to antibiotics during 90 days prior to isolation (85.4 %, 177/205), and the infections were mainly hospital-acquired (95.1%, 195/205) with only 4.9% from the community. The majority of MDR-PA isolates were resistant to cefepime (96.6%, 198/205), ciprofloxacin, piperacillin/tazobactam (91%, 186/205), and meropenem (90%, 184/205). Patient comorbidities with MDR-PA were diabetes mellitus (47.3%, n=97), malignancy (17.1%, n=35), end-stage renal disease (13.7%, n=28) and heart failure (10.7%, n=22). CONCLUSION: There was a significant prevalence of MDR-PA in Qatar, primarily from healthcare facilities and associated with prior antibiotic treatment. There was an alarming level of antimicrobial resistance to carbapenems. Our results are part of a national surveillance of MDR to establish effective containment plans.

Are adverse outcome rates higher in multiple sclerosis patients after total hip arthroplasty?

Aims: This study compared multiple sclerosis (MS) patients who underwent primary total hip arthroplasty (THA) with a matched cohort. Specifically, we evaluated: 1) implant survivorship; 2) functional outcomes (modified Harris Hip Scores (mHHS), Hip Disability and Osteoarthritis Outcome Score, Joint Replacement (HOOS JR), and modified Multiple Sclerosis Impact Scale (mMSIS) scores (with the MS cohort also evaluated based on the disease phenotype)); 3) physical therapy duration and return to function; 4) radiographic outcomes; and 5) complications. Patients and Methods: We reviewed our institution's database to identify MS patients who underwent THA between January 2008 and June 2016. A total of 34 MS patients (41 hips) were matched in a 1:2 ratio to a cohort of THA patients who did not have MS, based on age, body mass index (BMI), and Charlson/Deyo score. Patient records were reviewed for complications, and their functional outcomes and radiographs were reviewed at their most recent follow-up. Results: Compared with the matched cohort, MS patients had lower all-cause implant survivorship at eight years (91.5% (95% confidence interval (CI) 82.7 to 100) vs 98.7% (95% CI 96.2 to 100)) (p = 0.033), lower mHHS scores (66 vs 80, p < 0.001), and HOOS JR scores (79 vs 88, p = 0.009). Multiple sclerosis patients also required more physiotherapy (five weeks vs three weeks, p = 0.002) and took longer to return to baseline (seven weeks vs five weeks, p = 0.010) than the matched cohort. Furthermore, MS patients had more complications than the non-MS patients (six vs zero, p < 0.001). The worse outcomes of the MS group can potentially be explained by predisposition of these patients to mechanical complications and progression of their disease during the period of this study, as demonstrated by worsening of the mMSIS scores (2.9 vs 3.4; p = 0.008). Conclusion: MS patients had lower implant survivorship, lower functional outcome scores, and increased complication rates; in addition, MS patients took longer to return to their baseline functional level after THA. Cite this article: Bone Joint J 2018;100-B:875-81.

Peripartum hysterectomy: an economic analysis of direct healthcare costs using routinely collected data.

OBJECTIVE: To estimate resource use and costs associated with peripartum hysterectomy for the English National Health Service. DESIGN/SETTING: Analysis of linked Clinical Practice Research Datalink and Hospital Episodes Statistics (CPRD-HES) data. POPULATION: Women undergoing peripartum hysterectomy between 1997 and 2013 and matched controls. METHODS: Inverse probability weighted generalised estimating equations were used to model the non-linear trend in healthcare service use and costs over time, accounting for missing data, adjusting for maternal age, body mass index, delivery year, smoking and socio-economic indicators. MAIN OUTCOME MEASURES: Primary care, hospital outpatient and inpatient attendances and costs (UK 2015 prices). RESULTS: The study sample included 1362 women (192 cases and 1170 controls) who gave birth between 1997 and 2013; 1088 (153 cases and 935 controls) of these were deliveries between 2003 and 2013 when all categories of hospital resource use were available. Based on the 2003-2013 delivery cohort, peripartum hysterectomy was associated with a mean adjusted additional total cost of £5380 (95% CI £4436-6687) and a cost ratio of 1.76 (95% CI 1.61-1.98) over 5 years of follow up compared with controls. Inpatient costs, mostly incurred during the first year following surgery, accounted for 78% excluding or 92% including delivery-related costs. CONCLUSION: Peripartum hysterectomy is associated with increased healthcare costs driven largely by increased post-surgery hospitalisation rates. To reduce healthcare costs and improve outcomes for women who undergo hysterectomy, interventions that reduce avoidable repeat hospitalisations following surgery such as providing active follow up, treatment and support in the community should be considered. TWEETABLE ABSTRACT: A large amount of NHS data on peripartum hysterectomy suggests active community follow up could reduce costs, #HealthEconomics.

Antifolate drug resistance: Novel mutations and haplotype distribution in dhps and dhfr from Northeast India.

Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently the partner drug for artemisinin combination therapy (ACT) was sulphadoxine pyrimethamine (SP). Antifolate drug resistance has been associated with the mutations at dihydropteroate synthase (dhps) and dihydrofolatereductase (dhfr) genes. This study investigated antifolate drug resistance at the molecular level. A total of 249 fever cases from Arunachal Pradesh, NE India, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum. Samples were sequenced and analysed with the help of BioEdit and ClustalW. Three novel point mutations were found in the dhps gene with 10 haplotypes along with the already reported mutations. A single haplotype having quadruple mutation was found in the dhfr gene. The study reports higher degree of antifolate drug resistance as evidenced by the presence of multiple point mutations in dhps and dhfr genes. The findings of this study strongly discourage the use SP as a partner drug in ACT.

Human papillomavirus genotypes and HPV-16 variants distribution among Tunisian women with normal cytology and squamous intraepithelial lesions.

BACKGROUND: Little is known about the epidemiological characteristics of papillomavirus (HPV) infection among North African countries. Herein, we conducted a molecular epidemiological study to investigate prevalence of HPV type and HPV-16 variants among cervical-screened unvaccinated Tunisian women. METHODS: Cross-sectional study was performed on 494 Tunisian women visiting Women's Healthcare Centers. HPV-DNA detection was carried out on cervical samples using real-time polymerase chain reaction. HPV genotyping and HPV-16 variants were characterized by direct sequencing of L1 viral capsid gene. RESULTS: The overall HPV prevalence was 34% (95% CI: 30-38%) with significantly higher prevalence among women with squamous intraepithelial lesions (SIL) than those with no intraepithelial lesions (NIL) 84% (95% CI: 76-92%) and 24.5% (95% CI: 20-29%) respectively. The distribution of HPV prevalence according to women's age shows a U-shaped curve and the highest HPV prevalence rates were observed among the youngest (≤25 years; 51.2%, 95% CI: 37-67%) and the oldest women (>55 years; 41.7%, 95% The HPV-16 prevalence was 32.8% (95% CI: 22-45%) among women with SIL and 9.2% (95% CI: 6-12%) among women with NIL. Whereas, the HPV-18 prevalence was 1.3% (95% CI: 0-5%) among women with SIL and 0.3% (95% CI: 0-1%) among women with NIL. Among HPV-16 positive women, European lineage (E) was identified as the predominant HPV-16 variant (85.7%, 95% CI: 76-95%). The frequency of E variant was lower among SIL than among NIL women (81%, 95% CI: 64-99%, and 88%, 95% CI: 77-100%, respectively). Conversely, the African-2 variant frequency was higher among SIL than among NIL women (18%, 95% CI: 1-36% and 6%, 95% CI: 2-14%, respectively). In multivariate analysis, young age was the only risk factor that is independently associated with HPV infection. Moreover, HPV infection and menopause were both found to be independently associated with SIL and HSIL. CONCLUSION: HPV DNA testing should be proposed to young and menopausal Tunisian women. Considering HPV prevalence, only 13% of the Tunisian women could be protected by the bivalent HPV vaccine. These results may be helpful for designing an adapted HPV testing and vaccination program in Tunisia.

Risk of symptomatic venous thromboembolism following emergency appendicectomy in adults.

BACKGROUND: Appendicectomy is the commonest intra-abdominal emergency surgical procedure, and little is known regarding the magnitude and timing of the risk of venous thromboembolism (VTE) after surgery. This study aimed to determine absolute and relative rates of symptomatic VTE following emergency appendicectomy. METHODS: A cohort study was undertaken using linked primary (Clinical Practice Research Datalink) and secondary (Hospital Episode Statistics) care data of patients who had undergone emergency appendicectomy from 2001 to 2011. Crude rates and adjusted incidence rate ratios (IRRs) for VTE were calculated using Poisson regression, compared with baseline risk in the year before appendicectomy. RESULTS: A total of 13 441 patients were identified, of whom 56 (0·4 per cent) had a VTE in the first year after surgery. The absolute rate of VTE was highest during the in-hospital period, with a rate of 91·29 per 1000 person-years, which was greatest in those with a length of stay of 7 days or more (267·12 per 1000 person-years). This risk remained high after discharge, with a 19·1- and 6·6-fold increased risk of VTE in the first and second months respectively after discharge, compared with the year before appendicectomy (adjusted IRR: month 1, 19·09 (95 per cent c.i. 9·56 to 38·12); month 2, 6·56 (2·62 to 16·44)). CONCLUSION: The risk of symptomatic VTE following appendicectomy is relatively high during the in-hospital admission and remains increased after discharge. Trials of extended thromboprophylaxis are warranted in patients at particularly high risk.

Complementation of Plasmodium berghei TRAP knockout parasites using human dihydrofolate reductase gene as a selectable marker.

Previously we have used the Plasmodium dihydrofolate reductase thymidylate synthase (DHFR-TS) selectable marker to generate Plasmodium berghei TRAP null mutant parasites. These TRAP null mutants do not glide and they showed a great reduction in their ability to infect mosquito salivary glands and the hepatocytes of the vertebrate host. Thus far, complementation of these knockout parasites was not possible due to the lack of additional selectable markers. Recently, a new selectable marker, based on the human dihydrofolate reductase (hDHFR) gene, has been developed which confers resistance to the antifolate drug WR99210. This drug has been found to be highly active against pyrimethamine-sensitive and -resistant strains of P. berghei. In this study, we have used the hDHFR gene as a second selectable marker for the complementation of P. berghei TRAP null mutant parasites. Restoration of the TRAP null mutant parasites to the wild-type phenotype was achieved in this study via autonomously replicating episomes bearing a wild-type copy of the TRAP gene. This is the first report of complementation of a mutant phenotype in malaria parasites.

Green fluorescent protein as a marker in Plasmodium berghei transformation.

We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.

Subtle mutagenesis by ends-in recombination in malaria parasites.

The recent advent of gene-targeting techniques in malaria (Plasmodium) parasites provides the means for introducing subtle mutations into their genome. Here, we used the TRAP gene of Plasmodium berghei as a target to test whether an ends-in strategy, i.e., targeting plasmids of the insertion type, may be suitable for subtle mutagenesis. We analyzed the recombinant loci generated by insertion of linear plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that plasmid integration occurs via a double-strand gap repair mechanism. Although sequence heterologies located close (less than 450 bp) to the initial double-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated with plasmid integration into TRAP suggests that an ends-in strategy may be widely applicable to modify plasmodial genes and perform structure-function analyses of their important products.

Molecular mechanisms of malaria sporozoite motility and invasion of host cells.

Malaria sporozoites have the unique capacity to invade two entirely different types of target cell in the mosquito vector and the vertebrate host during the course of the parasite's life cycle. Although little is known about the specific interaction of the sporozoite with its target cells, two sporozoite proteins, circumsporozoite (CS) and thrombospondin-related adhesive protein (TRAP), have been shown to play important roles in the invasion of both cell types. CS protein is a multifunctional protein involved in sporogony, invasion of the salivary glands, the specific arrest of sporozoites in the liver sinusoid, gliding motility of the sporozoite, and hepatocyte recognition and entry. TRAP has been shown to be critical for sporozoite infection of the mosquito salivary glands and liver cells, and is essential for sporozoite gliding motility. This review will focus on the involvement of these molecules in sporozoite motility and the invasion of host cells.

TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites.

Many protozoans of the phylum Apicomplexa are invasive parasites that exhibit a substrate-dependent gliding motility. Plasmodium (malaria) sporozoites, the stage of the parasite that invades the salivary glands of the mosquito vector and the liver of the vertebrate host, express a surface protein called thrombospondin-related anonymous protein (TRAP) that has homologs in other Apicomplexa. By gene targeting in a rodent Plasmodium, we demonstrate that TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporozoite gliding motility in vitro. This suggests that in Plasmodium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.

Circumsporozoite protein is required for development of malaria sporozoites in mosquitoes.

Malaria parasites undergo a sporogonic cycle in the mosquito vector. Sporozoites, the form of the parasite injected into the host during a bloodmeal, develop inside oocysts in the insect midgut, then migrate to and eventually invade the salivary glands. The circumsporozoite protein (CS), one of the major proteins synthesized by salivary gland sporozoites, is a surface-associated molecule which is important in sporozoite infectivity to the host. Here, by gene targeting, we created Plasmodium berghei lines in which the single-copy CS gene was disrupted. The CS(-) and wild-type parasites produced similar numbers of oocysts of comparable size in the mosquito midgut. In the CS(-) oocysts, however, sporozoite formation was profoundly inhibited. CS therefore appears to have a pleiotropic role and to be vital for malaria parasites in both the vector and the host: in mosquitoes, CS is essential for sporozoite development within oocysts, and in the vertebrate host it promotes sporozoite attachment to hepatocytes.

Detection of Herpes Simplex Virus and candida infection with special emphasis on their possible role in human Squamous Cell Carcinoma and its variants.

Sixty seven cases of human oral S.C.C. with its intra oral variances were investigated microbiologically, histopathologically, histochemically and immunologically. Yeasts were isolated from 85% of examined cases. "Candifast test" showed that Candida albicans was the commonest form of the detected fungi, followed by C. parapsilosis and C. tropicalis, while the least in frequency was the Torulopsis glabrata. Yeasts were more detected in females, elders and tobacco smokers. Two cases of well differentiated S.C.C. were surprisingly detected, and for the first time in literature, associated with a specific granulomatous reaction and showed positively impregnated fungi. While negative reactions for fungi were noticed in all lymphoepitheliomas, almost all the verrucous carcinoma were positive. Viral inclusion bodies were demonstrated for the first time by MT. This method was rapid, economic and could be used as a pilot study before applying the more specific monoclonal antibody techniques or in-situ hybridization methods. Comparing the results of HSV hybridization with that of candida infection we found that both could be detected in some cases. The results indicate that viral and fungal factors may be synergetic in the development of oral carcinomas.

Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.

On the basis of conserved sequences characteristic of the Ran/TC4 subfamily of the GTPase superfamily, a fragment of the gene encoding a Plasmodium falciparum Ran/TC4 homologue was amplified in the polymerase chain reaction. The fragment was used to screen a cDNA library to obtain clones which allowed determination of the complete gene sequence. The gene, designated pfran (Plasmodium falciparum ras-like nuclear protein), has around 70% amino acid identity with previously characterised Ran/TC4 proteins. Like other malarial mRNAs, the pfran mRNA contains a long (at least 679 bp) 5' untranslated region. Southern blotting experiments show that pfran is a single copy gene located on chromosome 11. RNA hybridisation experiments indicate that pfran mRNA is abundant in late trophozoite and schizont stages, but present at very low levels in gametocytes and early asexual stages.

MVR-PCR analysis of hypervariable DNA sequence variation.

Techniques for accurate marking of infectious microbial agents circulating in populations would be very useful to epidemiologists. In this article, David Arnot, Cally Roper and Ali Sultan review recent progress in transferring MVR-PCR DNA finger-printing techniques from human forensic medicine to parasitology.